West Nile (WN) and dengue (DEN) viruses are emerging flaviviruses and potential biodefense pathogens that cause significant human diseases. Neither vaccine nor effective chemotherapy is currently available for WN and DEN infections in humans. The objective of this project is to identify novel inhibitors of WN and DEN viruses. We will accomplish the following two interim objectives. (1) Develop novel high-throughput assays to screen for inhibitors of WN and DEN replication. Using subgenomic WN and DEN replicons expressing reporter genes, we will develop reporting cell lines that can be used for high-throughput screening of inhibitors against all targets involved in viral replication. As a proof-of-principle, we have already established such a reporting cell line for high-throughput screening of WN inhibitors. We will develop a similar high-throughput assay for anti-DEN drug discovery. (2) Identify new classes of inhibitors of WN and DEN and analyze their modes of action. In collaboration with ViroPharma, Inc., we will use the high-throughput assays to identify new classes of WN and DEN inhibitors through screening a large number of compound libraries. We will study the potential inhibitors by (i) examining whether the compounds can inhibit other members of the flaviviruses, and (ii) analyzing the modes of action of the compounds through both biochemical (enzyme assays) and genetic approaches (selection of compound-resistant viruses). The high-throughput assay-approach proposed in this application has been proven to be fruitful in antiviral drug discovery (e.g. hepatitis C virus). At the completion of these studies, we expect to have identified novel inhibitors of WN and DEN, and to have determined the modes of action of these inhibitors. These results will be significant, because they will form a foundation for development of an efficacious chemotherapy of WN and DEN infections. The reporting cell lines developed in this project will also be useful for many aspects of flavivirus research such as studying genome packaging and viral particle formation.